Mutations in Aristolochene Synthase Promote Hydroxylation of Aristolochene in Aspergillus oryzae

A terpene synthase gene (mtas) from Menisporopsis theobromae BCC 4162 was heterologously expressed in Aspergillus oryzae NSAR1, resulting in the production of (+)‐aristolochene. Mutations were introduced in MtAS at aromatic residues (Y83, F103, F169, and W323) surrounding the active site, which are critical for precursor cyclisation and intermediate stabilisation during aristolochene biosynthesis. Transformants harbouring mutated mtas, specifically F103W, F169A and F169W, produced (2R,4S,5R,7S)‐2‐hydroxyaristolochene as the major product, along with aristolochene and other tentative metabolites, including germacrene A and sesquiterpenoids. Feeding experiments with (+)‐aristolochene in A. oryzae NSAR1 revealed that 2‐hydroxyaristolochene was only detected in trace amounts. This suggests that the oxidation products observed in this study are predominantly catalysed or facilitated by the mutated MtAS, differing from in vitro studies of other mutated aristolochene synthases. These findings highlight the impact of enzyme mutations on sesquiterpene production and suggest potential strategies for enhancing the diversity of sesquiterpenoids.